Cryptosporidium parvum is a well-recognized cause of diarrhea in humans and animals throughout the world, and is associated with a substantial degree of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). At the present time, there is no effective therapy for treating or preventing infection with C. parvum. This is primarily due to a lack of understanding of the basic cellular and molecular biology of this pathogen in terms of virulence factors, genome structure, gene expression, and gene regulation. With the recent advances in high-throughput automated DNA sequencing capabilities, large-scale genomic sequencing has become a cost-effective and time-efficient approach to understanding the biology of an organism. This is in stark contrast to the cost and time associated with single gene studies. Our preliminary studies on the genetic structure and genome organization of C. parvum have led us to formulate the hypothesis that genome sequencing will prove to be an efficient and cost-effective method for gene discovery for this eukaryotic pathogen. The following specific aims are proposed: 1) Generate and "end sequence" a random small-insert C. parvum genomic library; 2) Generate and "end sequence" large-insert (15-20 kb) C. parvum genomic libraries to provide a scaffold for sequence assembly; 3) Assemble sequences using Phred/Phrap/Consed software and assign contigs to specific chromosomes; 4) Finish sequencing of the genomic sequences; 5) Annotate/analyze the assembled completed genomic sequence to identify and characterize genes and structural features of the C. parvum genome. These studies will increase our basic understanding of the cellular and molecular biology of C. parvum in terms of gene and genome structure, and will identify key metabolic and pathophysiologic features of the organism for future development of sate and effective strategies for prevention and treatment of disease. Importantly, our preliminary data demonstrates that (a) we have all the necessary reagents and expertise required for completion of the proposed studies and (b) our ability to conduct large-scale analysis of the C. parvum genome.